Retinoids comprise a group of compounds including retinoic acid, retinol (vitamin A), retinal, retinyl acetate, retinyl palmitate, and natural and synthetic derivatives of these compounds, that together exert a profound effect on development and differentiation in a wide variety of systems. Although early studies focused on the effect of retinoids on growth and differentiation of epithelial cells, their actions have been shown to be widespread. Recent studies have examined the effects of retinoids on a variety of cultured neoplastic cell types, including the human promyelocytic leukemia cell line, HL60, where retinoic acid appears to be a potent inducer of granulocyte differentiation. In F9 embryonal carcinoma cells, retinoic acid will induce the differentiation of parietal endoderm, characteristic of a late mouse blastocyst. Retinoic acid also appears to play an important role in defining spatial-temporal axes in the developing avian limb and the regenerating amphibian limb.
Retinoic acid has been shown to induce the transcription of several cDNAs that have been isolated by differential screening, supporting the hypothesis that retinoic acid exerts its action via modulation of gene expression, mediated by a receptor protein.
The ability to identify compounds able to affect transcription of insect genes could be of significant value, as such compounds may be useful as insecticides. Of significance in this regard would be the identification of receptor proteins which modulate transcription of insect genes.
Through molecular cloning studies it has been possible to demonstrate that mammalian receptors for steroid, retinoid and thyroid hormones are all structurally related and comprise a superfamily of regulatory proteins that are capable of modulating specific gene expression in response to exposure to hormone, by binding directly to cis-acting elements [see, for example, Evans, Science 240:889 (1988); and Green and Chambon, Trends Genet. 4:309 (1988)]. Structural comparisons and functional studies with mutant receptors have made it possible to discern that these molecules are composed of a series of discrete functional domains, most notably, a DNA-binding domain that is composed typically of 66-68 amino acids, including two zinc fingers, and an associated carboxy terminal stretch of approximately 250 amino acids which comprises the ligand-binding domain (reviewed in Evans, supra).
An important advance in the characterization of this superfamily has been the delineation of a growing list of polypeptides encoded by DNA which has been isolated by low-stringency hybridization techniques using known receptor probes. These polypeptides possess the structural features of hormone receptors.
A retinoic acid dependent transcription factor, referred to as RAR-alpha (retinoic acid receptor-alpha), has been identified. Subsequently, two additional RAR-related genes were isolated, and there are now at least three different RAR subtypes (alpha, beta and gamma) known to exist in mice and humans. These retinoic acid receptors (RARs) share homology with steroid hormone and thyroid hormone receptors and have been shown to regulate specific gene expression by a similar, ligand-dependent mechanism (Umesono et al., Nature 336:262 (1988)). These RAR subtypes are expressed in distinct patterns throughout development and in the mature organism.
Recently, another retinoic acid-dependent transcription factor, designated RXR-alpha, has been identified in cDNA libraries prepared from human cells. RXR-alpha differs significantly in primary amino acid sequence from human RAR-alpha and other known members of the mammalian steroid/thyroid hormone superfamily of receptors; but, like other members of the superfamily, RXR-alpha is activated to effect transcription trans-activation in mammalian and insect cells exposed to retinoic acid and retinal. In addition, RXR-alpha effects trans-activation in mammalian cells exposed to a number of synthetic retinoids. The dose-response of RXR-alpha in CV-1 monkey kidney cells exposed to retinoic acid differs significantly from the dose response of human RAR-alpha exposed to the same conditions. See commonly owned, co-pending United States patent application Ser. No. 07/478,071, filed Feb. 9, 1990, now abandoned in favor of continuation-in-part U.S. Ser. No. 07/933,453, filed Aug. 21, 1992, now abandoned, which is incorporated herein by reference in its entirety.
The identification of yet other related receptors, especially from non-mammalian sources, would be of particular interest. Such discoveries would provide a great deal of information on the development of regulatory systems in the animal kingdom, and the potential to exert selective control over the proliferation of various pest species.
Other information helpful in the understanding and practice of the present invention can be found in commonly assigned, co-pending United States patent application Ser. Nos. 108,471, filed Oct. 20, 1987, issued Dec. 10, 1991, as U.S. Pat. No. 5,071,773; 276,536, filed Nov. 30, 1988, issued Jan. 1, 1991 as U.S. Pat. No. 4,981,784; 325,240, filed Mar. 17, 1989, now abandoned; 370,407, filed Jun. 22, 1989, now U.S. Pat. No. 5,516,322; and 438,757, filed Nov. 16, 1989, now pending, all of which are incorporated herein by reference in their entirety.